Abstract

Pneumolysin (PLY), an essential virulence factor of Streptococcus pneumoniae (pneumococcus), can penetrate the physical defenses of the host and possesses inflammatory properties. The vital role PLY plays in pneumococcus pathogenesis makes this virulence factor one of the most promising targets for the treatment of pneumococcal infection. Verbascoside (VBS) is an agent that does not exhibit bacteriostatic activity but has been shown to inhibit PLY-mediated cytotoxicity. The results from molecular dynamics simulations and mutational analysis indicated that VBS binds to the cleft between domains 3 and 4 of PLY, thereby blocking PLY’s oligomerization and counteracting its hemolytic activity. Moreover, VBS can effectively alleviate PLY-mediated human alveolar epithelial (A549) cell injury, and treatment with VBS provides significant protection against lung damage and reduces mortality in a pneumococcal pneumonia murine model. Our results demonstrate that VBS is a strong candidate as a novel therapeutic in the treatment of Streptococcus pneumoniae infection.

Introduction

Streptococcus pneumoniae (pneumococcus) infection causes significant morbidity and mortality around the world. S. pneumoniae causes a variety of diseases, ranging from otitis media to pneumonia, meningitis, and bacteremia, which have become increasingly difficult to cure. Drug-resistant strains exhibit resistance against β-lactam antibiotics and even novel antibiotics, such as vancomycin (Campbell and Silberman, 1998; Novak et al., 1999; Hidalgo et al., 2003). Pneumococcal pneumonia is a major cause of hospitalization. Pneumococcus is also one of the leading worldwide causes of deaths in children younger than 5 years, giving rise to an estimated 820,000 deaths in 2000 (Black et al., 2010).

In the articles of Paton et al. (1983, 1993), PLY, which is an indispensable virulence factor generated in the streptococcus pneumonia clinic isolation process, is a 53-kDa protein that belongs to the family of cholesterol-binding cytolysins (Palmer, 2001). Unlike other cytolysins, this hemolysin is located in the cytoplasm during the early period of bacterial growth and is released, due to growth and lysis, into the extracellular environment (Balachandran et al., 2001) where it localizes to the cell wall (Price and Camilli, 2009). Once this protein has localized to the cell wall, it binds to cholesterol in the host cell’s cytoplasmic membrane, infiltrates this membrane, and oligomerizes to form relatively large pores. During this process, the targeted cell is lysed as these pores are formed. In this respect, oligomerization is a key aspect of PLY’s mode of action. Similar to autolysin and surface protein A, vaccines against which have been proven to provide protection against S. pneumoniae infection (Musher et al., 2001; Wu et al., 2010), PLY is regarded as a pneumococcal protein vaccine candidate. PLY’s interaction with the cells of the respiratory system is the likely cause of alveolar edema and hemorrhage during the disease course. PLY also attenuates phagocyte and immune cell function, thus repressing host inflammatory and immune responses (Boulnois et al., 1991). In 2001, Jedrzejas (2001) pointed out that one important factor of S. pneumonia boarding on its host is the activity, especially the activity at the initially infected phase. Therefore, the discovery of inhibitors that target PLY could open novel therapeutic avenues for treating pneumonia infections.

Verbascoside (VBS) is a phenylpropanoid glycoside, and plants containing VBS are widely used in Chinese herbal medicine (Pu et al., 2003). Previous studies have shown that VBS has various pharmacological activities, including antioxidant activity (Chiou et al., 2004), hepatoprotective activity (Xiong et al., 1998), and anti-inflammatory and antinociceptive activities (Schapoval et al., 1998). VBS’s ability to affect hemolytic activity was identified via drug screening. Our present study showed that VBS, a natural product with no anti-S. pneumoniae activity, can lower the virulence of S. pneumoniae in vivo and in vitro by influencing the lytic activity of PLY, which indicates that VBS may interact directly with PLY. To determine the inhibition mechanism, standard molecular dynamics simulation, binding energy profiles, protein mutants, and principal component analysis were performed for the PLY-VBS complex. The results indicated that VBS can bind to the cleft between domains 3 and 4 of PLY, thereby blocking its oligomerization and inhibiting its lytic activity.

The results of this study may be useful for drug design against pneumococcal infection. Indeed, an antivirulence therapeutic strategy may be beneficial as an adjuvant to antibacterial therapy, because it is unlikely to accelerate antibacterial resistance (Wang et al., 2015).

Materials and Methods

Bacterial Strains and Culture.

S. pneumoniae strain D39 (NCTC 7466) and strain PLN (an isogenic pneumolysin-deficient S. pneumoniae) were selected for this study (Berry et al., 1989). The S. pneumoniae strains were cultured at 37°C using Todd-Hewitt broth. Calculation of minimal inhibitory concentration (MIC) of VBS for S. pneumonia is available when broth microdilution method is adopted and upon confirmation of the stipulations of the Clinical and Laboratory Standards Institute (CLSI). The minimal inhibitory concentrations (MICs) of VBS for S. pneumoniae were identified using the broth microdilution method based on the Clinical and Laboratory Standards Institute (CLSI) guidelines.

Construction, Expression, and Purification of PLY.

Primers were designed to amplify the S. pneumoniae gene encoding ply using S. pneumoniae strain D39 as the template. The expressions of the primers are listed in the following:

Forward 5′-CGCGGATCCGCGATGGCAAATAAAGCAGTAAA-3′ as well as the reverse 5′-CCGCTCGAGCGGCTAGTCATTTTCTACCTTAT-3′ (restriction enzyme sites are underlined). BamHI and XholI enzymes were used to digest the amplified ply gene, and the digested genes were then cloned into a pET28a prokaryotic expression vector. To overexpress the recombinant protein, the pET28a-PLY vector was transformed into Escherichia coli BL21 (DE3).

The cells were cultured until absorbance at 600 nm reached 0.6–0.8 at 37°C, induced with 0.3 mM IPTG, and harvested after growing for an additional 12 hours at 16°C. The harvested cells were resuspended in sterile PBS and crushed by a high-pressure homogenizer (EmulsiFlex-C3, AVESTIN, Ottawa, Ontario, Canada). The cell lysate was centrifuged at 12,000 g for 30 minutes, and the supernatant was loaded onto a Ni-NTA agarose column. The recombinant protein was bound to a Nickel-affinity chromatography column, and the nickel column was then flushed with washing buffer consisting of 20 mM Tris, 20 mM imidazole, and 300 mM NaCl (pH 8.0) The His-tagged protein was eluted in elution buffer containing 20 mM Tris, 300 mM imidazole, and 300 mM NaCl (pH 8.0). The eluted solution was concentrated using a Millipore Amicon filter (30 kDa molecular weight cutoff; Bedford, MA) for desalting. The purified product was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Hemolytic Test.

In 2010, Ragle and other researchers said that the assessment method of the hemolytic activities is as same as that used in other places (Ragle et al., 2010). Briefly, 10 μl of purified PLY was preincubated in a 96-well plate with a series of different concentrations of VBS in PBS at 37°C for 10 minutes. Defibrinated sheep blood erythrocytes (50 μl at 5 × 106 cells/ml) were added to the wells, and the final volume of the wells was then increased to 200 μl with PBS. Then, the samples were subject to incubation at the temperature of 37°C for 10 minutes. After centrifugation at 3000 g for 5 minutes, the supernatants were removed into a cuvette to measure their absorption at 543 nm.

Immunoblot Analysis.

S. pneumoniae were cultured at 37°C in Todd-Hewitt broth with various concentrations of VBS until the OD600 reached approximately 1.0. Equal volumes were boiled in Laemmli sample buffer. After being loaded into a 12% SDS-PAGE gel, the centrifuged supernatant samples were delivered to the polyvinylidene fluoride membranes through a half-dry carrier cell. The polyvinylidene fluoride membrane was then cut and sealed off for 2 hours with 5% skim milk at room temperature. The primary PLY antibody was added at a 1:1000 dilution and incubated overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse antiserum (secondary antibody) was diluted to 1:2000. The testing reagent for Amersham (Pittsburgh, PA) ECL immunoblotting was applied to develop the blots.

Site-Directed Mutagenesis.

Three amino-acid mutations (S254A, W278A, L447A) were created using a QuikChange site-directed mutagenesis kit. The template plasmid was the pET28a-PLY plasmid noted above. The primers used to mutate the relevant amino acids are shown in Table 1.

Oligomerization Analysis.

rPLY (5 nM at 10 mg/ml) was mixed with 10 nM VBS or 10 nM cholesterol liposome (CHO), 5 nM of rPLY at the same volume with neither agent was used as a control, and CHO (10 nM):VBS (20 nM) = 1:2 mixed with rPLY was used to attest the interference of VBS. After incubating at 37°C for 1 hour, the mixture and control sample were subjected to high-performance liquid chromatography (DGU-20A5, Shimadzu Corporation, Kyoto, Japan) using Nanofilm SEC-250 (Sepax Technologies, Inc., Newark, DE), wherein 0.5 ml the Nanofilm SEC-250 was filled per minute.

Live/Dead and Cytotoxic Testing.

The experimental cells and the alveolar epidermal cells, which were bought from ATCC (Manassas, VA), were cultured in the Dulbecco's modified Eagle medium, an expression vector according to Chemical Abstracts of the United States, and the content of fetal calf serum in the Dulbecco's modified Eagle medium is 10%. The cells were delivered to a 96-well plate, wherein each well can deliver 1.5 × 104 cells. The A549 cell was subjected to isolated incubation under assistance of S254A, WT-PLY, W278A, and L447A (0.5 μl, at the same concentration) and increasing concentrations of VBS. These test samples as well as positive control (PBS) were placed in a temperature-controlled box for 6 hours at 37°C. The LDH, namely the Cytotoxic Detection Kit (Roche Mannheim. Germany), can be adopted to measure releasing capacity of LDH, and the cellular activity could be determined. Live/dead assays were performed according to the methods recommended by the manufacturer (Invitrogen, Carlsbad, CA). LDH activity was measured on a microplate reader (TECAN, Salzburg, Austria). Microscopic images of stained cells were captured using a confocal laser scanning microscope (Olympus, Tokyo, Japan). The results are representative of a minimum of three independent experiments.

Pharmacokinetics Study.

Employing a protocol based on previous pharmacokinetic research methods (Li et al., 2014), analytes were separated on an Agilent Zorbax C18 column (Santa Clara, CA). As to the mobile phase, it contained solvent A (methyl alcohol) and solvent B (the content of methane acid in ddH2O being 0.1%). The flow rate was 0.8 ml/min. The injection volume was 10 μl, the detection wavelength was at 330 nm; meanwhile the blood temperature was kept at 25°C. Mouse blood samples were collected from veins into heparinized tubes at 5, 15, 30, 45, 60, 120, 240, and 360 minutes after dosing. Blank blood was collected before dosing.

Murine Model of Endonasal Pulmonary Infection.

The female C57BL/6J mice, which have grown for 8 weeks and weigh 20 ± 2 g, were bought in the Experimental Animal Centre of Jilin University located in Changchun City, Jilin Province. The mice used for tests rested for 1 week to acclimatize before use. The whole processes gained authorization and were implemented according to the stipulations of the ACUC (Animal Care and Use Committee) affiliated to the Jilin University.

The experimental methods used for inducing pneumonia in mice were presented previously (Dessing et al., 2008). The mice were lightly anesthetized by inhalation of isoflurane and then inoculated with 20 μl of suspension containing 5 × 107 colony-forming units (cfu) of strain D39 or strain PLN in the left nare. To test the effect of VBS treatment, several injections of 100 µl of VBS were subcutaneously administered to the mice 2 hours after infection with strain D39. Measurements were taken every 3 hours for a period of 120 hours. About 100 μl of aseptic poly butylenes succinate was filled in each of the mice under control.

Bronchoalveolar Lavages.

Bronchoalveolar lavage (BAL) fluid collections were administered twice by intratracheal instillation of 500 μl of sterile PBS. The clear surface liquid obtained after centrifugation was collected, and the lavage fluid supernatants were used for cytokine detection (Qiu et al., 2012).

Measurement of Bacterial Loads.

The bacterial numbers were identified by colony counts of lung tissue smears as previously described (Dessing et al., 2007). Lungs were collected from euthanized mice, and lung tissue homogenates were prepared in 1 ml of sterile PBS at 4°C and used to calculate the bacterial colony counts through the serial dilution method and smearing on solid media.

Homology Modeling Study.

The monomeric 3D structure of the pneumolysin (PLY) has not yet been reported. Therefore, a homology simulation study of PLY was performed using the structure of Perfringolysin O as the template (Protein Data Bank code 1M3I). The Align Sequence to Templates tool in Discovery Studio2.5 (Discovery Studio, BIOVIA, San Diego, CA) was used to carry out the sequence alignment between PLY and the template (Laskowski et al., 1993). The 3D structure of PLY was then constructed using the Build Homology Models protocol implemented in Discovery Studio 2.5. Subsequently, to obtain the equilibrium structure, a 500-ns molecular dynamics simulation was performed with the 3D structure of PLY using the Gromacs 4.5.5 software package (Hess et al., 2008). To achieve docking as well as dynamic analogy of molecules, the Gaussian 09 program was used to optimize the structure of VBS at the B3LYP/6-31G* level (Frisch et al., 2009).

Molecular Docking.

In this work, verbascoside (VBS), considered to be the flexible ligand, was docked into PLY using the docking program AutoDock 4.0 (AutoDock program, The Scripps Research Institute, La Jolla, CA) (Morris et al., 1996, 2009; Hu et al., 2009). The detailed docking process docking was reported previously (Dong et al., 2013). The docking model of VBS with PLY has been provided as the Supplemental Material.

Molecular Dynamics Simulation.

A molecular dynamics simulation of the complex of PLY with VBS was carried out using the Gromacs 4.5.5 package to explore the binding mode of complex. The Amberff99sb force field and TIP3P water model were applied (Ryckaert et al., 1977; Jorgensen et al., 1983). The specific parameters of the molecular dynamics (MD) simulation were consistent with those described previously. The force field parameters of VBS were estimated based on one portion of AM1-REST atom charges, which belongs to the program packages of Amber (Wang et al., 2006).

Principal Component Analysis.

Principal component analysis (PCA) is the simplest of the multivariate techniques that are used to reduce or simplify large, complex datasets (Zhou et al., 2001; Barrett and Noble, 2005; Liu et al., 2008). In this work, to define the dominant motion over an MD simulation, the collective motions of the complex were addressed by using the positional covariance matrix, Cα, of the atomic coordinates and its eigenvectors based on PCA. The Gromacs 4.5.5 module was used to perform PCA, and the trajectories were obtained from the previous MD simulations. The detailed process of principal component analysis was based on previous reports in the literature (Qiu et al., 2013).

Fluorescence-quenching Assay.

Verbascoside to the binding sites of wild-type and mutant PLY, namely the binding constants (KA) could be calculated in a fluorimetric quenching way. The binding energy of the complexes were converted from the binding constants using ΔGbind = RTlnKA (Qiu et al., 2012; Wang et al., 2015).

Statistics.

The 13.0 version Statistic Package for Social Science (SPSS Inc., Chicago, IL) was used to analyze the empirical data. Fisher type precision experiments were used to calculate the meanings of the mortality research statistically. Differences were considered statistically significant when P < 0.05.

Results

Inhibition of PLY-Induced Hemolytic Activity by Verbascoside.

Previous studies have shown that some extracts from traditional Chinese medicine have bacteriostatic activity against S. pneumoniae. Verbascoside (Fig. 1A) is a compound that occurs relatively widely in traditional Chinese medicine and has various pharmacological activities. Studies examining the MIC found that even a concentration of VBS as high as 2048 μg/ml was still not capable of impeding the growth of the bacteria, indicating that VBS exhibits no antimicrobial activity against S. pneumoniae. However, VBS was found to have a negative influence on the hemolytic activities of PLY on the basis of different concentrations via drug screening (Fig. 1, B and C). Furthermore, VBS does not affect the production of PLY in S. pneumoniae (Fig. 1D). The above results implied that VBS may directly interact with PLY.

Suppressing effect of verbascoside (VBS) on hemolysis caused by pneumolysin (PLY). (A) Chemical structural formula of VBS. (B and C) As determined through hemolysis tests using purified rPLY and sheep blood erythrocytes in PBS and measuring the absorbance values of the centrifugal supernatants at 543 nm, the addition of VBS decreased the hemolysis rates. The column diagrams stand for the average values of the assays (n = 3). The error bars stand for standard errors. **When contrasting to the matched group, P is smaller than 0.01. (D) Western blot analysis of PLY expression. Bacteria lysate of S. pneumoniae D39 cultured in increasing concentrations of VBS. VBS is not able to influence PLY from being generated in S. pneumoniae. (E) Inhibition of PLY oligomerization by VBS. Purified rPLY that was able to self-associate was incubated with or without VBS at 37°C for 1 hour. Peak 1 presents PLY oligomers. Peak 2 and Peak 3 present PLY monomers. The results indicate that VBS interacts with PLY and makes the cytotoxin incapable of self-association. F, Peak 4 represents PLY oligomers, Peak 5 and Peak 6 represent PLY monomers, indicating that VBS could detectably interfere with CHO-induced Ply oligomerization.

Inhibition of PLY Oligomerization by VBS.

To find whether VBS weakens the hemolytic activity of PLY by inhibiting its oligomerization, we used size exclusion chromatography to separate PLY monomers and oligomers and monitored them by high-performance liquid chromatography. We already know that PLY in a solution that lacks cholesterol or membranes can self-associate to form oligomers from monomers (Gilbert et al., 1998). We mixed 10 nM of VBS into an rPLY sample and incubated the mixture at 37°C for 1 hour. Another rPLY sample without VBS was used as the control. Peak 1 represents PLY oligomers, whereas Peak 2 and Peak 3 represent PLY monomers. The rPLY incubated with VBS could not form oligomers and exhibited only a monomer peak (Peak 3) (Fig. 1E); thus, the PLY in this sample was incapable of self-association. Cholesterol (CHO) is the natural ligand of PLY and it induces oligomerization of the protein. We mixed 10 nM CHO into an rPLY sample, and mixed CHO (10 nM):VBS (20 nM) with rPLY to attest the interference of VBS. Peak 4 represents PLY oligomers, Peak 5 and Peak 6 represent PLY monomers (Fig. 1F). The results indicate that VBS could detectably interfere with CHO-induced Ply oligomerization. From what has been shown above, we could safely draw the conclusion that VBS molecule could inhibit PLY oligomerization.

Identification of the Binding Mode of PLY with VBS.

To obtain the stable structure of PLY in complex with VBS, standard MD simulations were performed for the complexes, and the root-mean-square deviations of backbone Cα atoms were used provide the stability and conformational drift of PLY in the simulation. As shown in Fig. 2A, the protein in the complex system could reach the plateau at 20 ns at a value of ∼0.35 nm with small fluctuations at the value of ∼0.1 nm. However, the protein in the free protein system could also reach the plateau at 20 ns with the bigger fluctuations around the value of ∼0.2 nm due to the binding of PLY with VBS.

The dynamic properties of PLY with VBS complex. (A) The root mean square deviation values of PLY with VBS complex (black line) and unliganded PLY (red line) via simulation times; (B) the number of H-bonds in the complex system fluctuates within the simulation time; (C) the root mean square fluctuation (RMSF) of the residues of PLY in the complex and unliganded protein was calculated during the last 100-ns simulation.

The stable structure of PLY with VBS was obtained via the 200-ns MD simulation, as shown in Fig. 3. VBS could be precisely embedded into the cleft between domains 3 and 4 in PLY through the H-bonding as well as hydrophobic interactions. The hydroxyl group of the benzene ring on the right side of VBS can form a strong hydrogen bond with Asp471, which has a crucial significance in making the right side of VBS to be stable. Moreover, two >O atoms in the central section of VBS can form three hydrogen bonds with Asn470, and the hydroxyl group can form a hydrogen bond with Glu277, indicating that the central section of VBS can be anchored by Asn470 and Glu277. The quantity of H-bonds that were found between PLY and VBS during the last 160-ns simulation was displayed in Fig. 2B, fluctuating from 4 to 6 within the simulation time, which is consistent with the above results. To ensure the effectiveness of hydrogen bonds between VBS and PLY, the stability of the hydrogen bonding was calculated in Table 2, which confirms that the hydrogen bonds can exist in a stable manner. In addition, the plane of the benzene ring on the left side of VBS and the plane of benzene rings of the remained Tyr358 are level to each other. Thus, there is likely a strong π-π interaction between this residue and VBS, stabilizing the left side of VBS in complex with PLY.

The binding mode of VBS with PLY on the basis of MD simulation. During the standard 200-ns MD simulation, VBS could strongly bind at the cleft between domain 3 and domain 4 in PLY. Residues Asp471, Asn470, Glu277, Tyr358, and Arg359 play key roles in VBS binding with PLY.

To further validate the binding sites of PLY with VBS and to study the flexible property of the remained Tyr358, the root mean square fluctuation (RMSF) value, namely the root mean square fluctuation value of the remained Tyr358 that enclosed the bonding positions of PLY was computed. As shown in Fig. 2C, the flexibilities of the residues surrounding the binding sites in the free protein and complex are different. The residues (250–471) of PLY that bind VBS show weaker flexibility when bound to VBS, with a RMSF value of less than 0.40 nm, compared with the corresponding residues in the free protein. This finding indicates that these residues are more rigid due to binding with VBS. Given the above information, the stabilization at the binding site of PLY with VBS was largely due to residues Asp471, Asn470, Glu277, Tyr358, and Arg359, as shown in Fig. 3.

Binding Energy Calculation of VBS to PLY.

The initial binding mode of PLY with VBS could be identified by the 3D structure based on the MD simulation. However, the information obtained for the binding site residues in the complex is not sufficient. To address this problem, the importance of remained binding sites for the bonding energy between PLY and verbascoside could be assessed in an MM-PBSA way, and the sums of the bonding energy of every remained binding spot could be partitioned into the electrostatic (ΔEele), the salvation (ΔEsol), the Van der Waals (ΔEvdw) and the total contribution (ΔEtotal), as shown in Fig. 4. As shown in Fig. 4A, although the value of Glu277 is smaller than −2.0 kcal/mol, its electrostatic (ΔEele) contribution is great, indicating a strong electrostatic interaction between PLY and the middle part of VBS. However, because of the unfavorable contribution of solvation (ΔEsol), with a value of ∼2.0 kcal/mol, the total contribution (ΔEtotal) of Glu277 is low, with a value of ∼−0.8 kcal/mol. Consistent with the results of the above analysis, Asn470 and Asp471 have strong electrostatic interactions with VBS, with values of ∼−2.31 and −0.96 kcal/mol, respectively, because of the formation of hydrogen bonds. Moreover, as shown in Fig. 4A, Tyr358 and Arg359 have strong Van der Waals terms of ∼−2.0 and ∼2.3 kcal/mol (ΔEtotal), respectively. In summary, this analysis confirms that the key residues of the binding site are Asp471, Asn470, Glu277, Tyr358, and Arg359.

The binding free energy contribution of residues in the PLY binding sites. The binding energies decomposition based on a per-residue at the binding sites among WT-PLY (A), S254A-PLY (B), W278A-PLY (C), and L447A-PLY (D) and VBS. The bar graph illustrates the van der Waals (white), electrostatic (blue), salvation (yellow), and total (black) contributions for the complexes.

To confirm the binding site in the PLY-VBS complex, similar MD simulations were performed for the complexes containing S254A-PLY, W278A-PLY, and L447A-PLY mutants bound to VBS, and then the binding free energies of the three complexes were calculated using the MM-PBSA method. Then, a method of fluorescence spectrum quenching was used to calculate the binding free energies of VBS with the three mutants. As shown in Fig. 4, B, C, and D, the key residues in the binding site of complexes are similar to those of WT-PLY in complex with VBS, except for the mutant residues. The total binding free energy of WT-PLY, S254A-PLY, W278A-PLY, and L447A-PLY in complex with VBS and their energy contributions are summarized in Table 3. It can be seen from the calculation of the binding free energy of the complex that in comparison with the WT-PLY/VBS complex, the value of the binding free energy was reduced by about 4–5 kcal/mol. According to the results based on the fluorescence spectroscopy quenching method, the binding free energy between VBS and PLY decreases in the following order: WT > S254A-PLY > W278A-PLY > L447A-PLY, well consisting with the results of the MD simulation. These analyses provide further evidence that the binding of PLY to VBS is due to the residues Asp471, Asn470, Glu277, Tyr358, and Arg359.

The binding free energy of VBS binding with WT-PLY and mutants based on the calculations, and the binding constants (KA) (1 × 105) l⋅mol−1 of the PLY-VBS complexes systems gained from the fluorescence-quenching method

Principal Component Analysis of the Motion for PLY from the Complex.

According to previous reports, the hemolytic activity of PLY can be achieved by monomeric oligomerization resulting from a conformational change of the PLY monomer. MD simulation and hemolysis experiments show that as VBS directly interacts with protein, it has a negative effect in the hemolytic activities of PLY. Therefore, this paper discusses the most important motions of PLY with a ligand or without it so as to seek the inhibitory mechanism of VBS by principal component analysis (PCA) on the basis of the molecular dynamics tracks of PLY-VBS complexes and unbound PLY. As shown in Fig. 5, there is an extended motion between domains 3 and 4, which has an overall influence on the structure of the isolated protein in a first element (PC1), wherein the dash line in Fig. 5 stands for PC1. In addition, the extended motion is sufficiently large that PLY can successfully change from the monomer structure to the oligomeric structure. A slight vibration of the backbone of the protein was shown in the second principal component (PC2), as shown in Fig. 5. Interestingly, VBS binds in the cleft between domains 3 and 4 of PLY based on the MD simulation, indicating that the motion of domains 3 and 4 can be influenced by the binding of VBS. As expected, the extended motion between domains 3 and 4 is clearly weaker in the PLY/VBS complex compared with that of the unliganded PLY, as shown in Fig. 5. Thus, this analysis confirmed that the motion of the conformation transition for PLY from the monomeric to the oligomeric form is restricted by the binding of VBS with PLY.

Principal component analysis of the PLY motion. The first and second principal components (PC1 and PC2) in free protein (A) and the first and second principal components (PC1 and PC2) in complex (B) obtained by PCA are depicted by cones on the alpha carbon atoms. The length of the cones represents the magnitude of the motion. The dotted line range represents the binding region of VBS with PLY.

Moreover, to further confirm the above mechanism, the distances between domains 3 and 4 were calculated in PLY with VBS and without VBS system, as shown in Fig. 6. The average distances for the PLY/VBS complex and free PLY were 4.25 and 4.50 nm, respectively. These results indicate that the conformation of domains 3 and 4 in PLY was restrained due to the binding of VBS, which is consistent with the above results.

The distance between the domain 3 and domain 4 as a function of time. The red and black lines represent the distance between domain 3 and domain 4 in unliganded PLY and the complex of PLY-VBS, respectively.

In summary, based on these findings, the inhibition mechanism can be hypothesized: the conformational change from the monomeric to oligomeric form is blocked due to the binding of VBS to the cleft between domains 3 and 4 in PLY, leading to a decrease in the lytic activity of PLY.

It has long been known that PLY, a pore-forming cytotoxin of S. pneumoniae, directly contributes to alveolar epithelial and pulmonary endothelial injuries (Jedrzejas, 2001). In the past, how S. pneumoniae injures lung cells was determined under assistance of the lung alveolar epithelium cell of people, represented by A549 (Schmeck et al., 2004), and PLY has been found to be the major factor involved in their apoptosis and necrosis (Jedrzejas, 2001). As shown in Fig. 7, it can be seen that the intact A549 cells were detected retained a green fluorophore (Fig. 7A), whereas A549 alveolar epithelial cells, which were incubated together with PLY, contained the red fluorophore, indicating their dead status (Fig. 7B). In contrast, treatment with 8 μg/ml VBS protected A549 cells from death (Fig. 7C). The addition of 32 μg/ml VBS provided nearly complete protection to A549 cells (Fig. 7D). Moreover, a LDH release assay was performed to evaluate the effect of VBS on the PLY-mediated lysis of A549 cells. As expected, the addition of 2 to 32 μg/ml of VBS to samples reduced the cytotoxicity of WT-PLY in a dose-dependent manner (Fig. 7E). We created PLY variants with amino acid mutations at Ser254, Trp278, and Leu447; purified these mutants; and examined their effects in LDH release assays. The mutants did not have significantly altered cytotoxicity relative to WT-PLY. However, VBS did not produce conspicuous cytoprotective effects when samples containing mutant PLY were treated with various concentrations of VBS (Fig. 7, F-H). These dramatic results confirm our hypothesis that VBS exhibits a potential therapeutic effect that merits further investigation and indicate that as we predicted, mutations in the aforementioned residues impair the effectiveness of VBS.

VBS alleviates PLY-mediated A549 cell injury. When VBS was treated in the environment of pneumolysin for 6 hours, the live pictures in green color or the dead pictures in red color of A549 cells could be acquired with help of the confocal laser scan microscope. (A) Untreated experimental cells. (B) A549 cells treated with PLY and without VBS treatment. (C and D) Cells cultured in the presence of 8 and 32 μg/ml VBS, respectively. The data in (A–D) are representative of three independent experiments. (E) VBS hinders the cytotoxicity of PLY. The LDH release by the experimental cells was measured after treatment with PLY in the presence of the indicated VBS concentrations. (F–H) PLY variants with mutations at Ser254, Trp278, and Leu447 did not exhibit marked changes in activity relative to WT-PLY; however, the addition of different concentrations of VBS to samples with PLY mutants did not produce conspicuous cytoprotective effects. *P < 0.05 and **P < 0.01, according to two-tailed Student’s t tests.

VBS Protects Mice from S. pneumoniae Pneumonia.

Based on the above-presented experimental data, we sought to analyze whether similar protection would occur in vivo in mouse affected with pneumonia caused by S. pneumoniae. The first step was to analyze the pharmacokinetic features of VBS in mice. After subcutaneous injection of a single VBS dose of 100 mg/kg, we detected the drug concentrations in the mice plasma. The maximum concentration (Cmax) of VBS in plasma was 112.22 μg/ml 30 minutes after administration (Fig. 8A), suggesting that VBS effectively enters the systemic circulation.

Verbascoside (VBS) protects mice against Streptococcus pneumoniae pneumonia. (A) Pharmacokinetics of VBS. The female C57BL/6J mice received a single subcutaneous dose of 100 mg/kg. Mouse eyeball blood samples were collected at designated times to determine the serum concentrations of VBS. (B) The influence on the mortality of the mice that were infected. Approximately 5 × 107 cfu of strain D39 were suspended in the left nare of C57BL/6J mice, and the mortality of mice was supervised for 120 hours. D39-infected mice were treated with VBS 2 hours after infection. The other mice injected with sterile PBS were influenced with D39 and subjected to PLN formed the matched groups. (C) The influence of VBS exerted on the lung bacteria burden of infected mice. C57BL/6J mice were administered 5 × 107 bacteria of the above-mentioned strains. The infected extent could be estimated by accounting the quantity of the lung bacteria of the mice that had been influenced for 48 hours. (D–E) Pathologic (D) and histopathological changes (E) in the lung tissue from S. pneumoniae-infected mice (48 hours after infection).The lung tissues were dyed with hematoxylin and eosin (original magnification, ×400). (F–G) Influence of VBS on inflammatory factors in infected mice. Tumor necrosis factor α (TNF-α; F) and interleukin 1β (IL-1β; G) were assessed in the BAL fluid of mice (48 hours after infection). The experimental results shown in (B–G) are from three separate tests, wherein *P < 0.5 and **P < 0.01. Kaplan-Meier tests were used to assess the survivorship curves, and two-tailed Student’s t tests were adopted to analyze the remaining experimental data.

Mortality due to pneumonia caused by S. pneumoniae D39 was monitored over a 120-hour time course. Approximately 75% of C57BL/6J mice infected with S. pneumoniae were dead within 120 hours. Through a test where the mice were treated with 100 mg/kg VBS, it can be seen that an obvious protection played a role, with a mortality rate 120 hours after infection of only 25%. As expected, none of the mice infected with strain PLN (the isogenic pneumolysin-deficient S. pneumoniae) died within the time course (Fig. 8B). Obviously, the survival time of the VBS-treated group was longer than that of the untreated group (P < 0.01). Additionally, the bacterial count in the lungs was significantly lower in the VBS-treated group than in the PBS treated group (P < 0.01), indicating that VBS influences S. pneumoniae survival within the lungs (Fig. 8C).

Through the macroscopic inspection, it can be seen that the untreated mice had dark red lungs that exhibited severe congestion, whereas the lung tissue of mice treated with VBS and infected with the PLN strain was pink and fungous (Fig. 8D). Our examination of the pathologic manifestations revealed that the S. pneumoniae-infected mice in the PBS group exhibited severe tissue injury and an accumulation of inflammatory cells in the alveolar space. In contrast, the pathologic tissue sections of the mice in the treatment group were similar to those of the mice infected with the PLN strain. The histopathological features corresponded to a relief of pulmonary inflammation, as suggested by a reduction of inflammatory exudates (Fig. 8E). Furthermore, we analyzed the levels of various cytokines, including tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), in the bronchoalveolar lavage (BAL) fluid of the experimental mice. Consistent with our previous observations, the concentrations of TNF-α and IL-1β were significantly lower in VBS-treated mice than those observed in the control mice (P < 0.05) (Fig. 8, F and G). Taken as a whole, these results demonstrated that VBS treats S. pneumoniae pneumonia in a murine model of infection.

Discussion

Conventional antibiotics are always recommended as the primary therapy for S. pneumoniae-infected patients. Although these traditional approaches are effective, they have generated evolutionary stress on the target bacterium, which has thus been selected for resistant subpopulations (Werner et al., 2008). The emergence of antibiotic resistance and even the appearance of multidrug-resistant S. pneumoniae have made infectious diseases more difficult to cure and have increased the mortality rate (Thummeepak et al., 2015). Increasing understanding of and research into antivirulence has been pursued and has revealed a potential strategy to develop novel drugs to treat bacteria-mediated disease (Rasko and Sperandio, 2010). Instead of applying conventional antibiotics to exterminate microbes, this approach might present a moderate selection pressure because it weakens the pathogen’s virulence and does not increase survival pressure.

Certain proteins trigger a pathogenic mechanism and participate in the disease progress induced by these organisms. The usability of PLY, one of these proteins, has been proposed (Jedrzejas, 2001). Here, we provide evidence that VBS has no antimicrobial activity against S. pneumoniae, but was found to antagonize formation of PLY’s oligomerization, thus weakening the hemolytic activity of PLY in a concentration-dependent way. Additionally, VBS hindered injury of pulmonary epithelial cells by PLY in an LDH release assay. PLY mutants produce stable cytotoxic effects that cannot be weakened by VBS, indicating that the mutated residues affect binding between PLY and VBS and thereby providing evidence for our molecular modeling results. Moreover, the agent obviously exerts protection in mouse model of pulmonary infection by alleviating lung pathologic damage and reducing mortality. In addition, VBS exhibited anti-inflammatory properties in VBS-treated mice, and this biologic effect may also play a role in alleviating lung injury.

For the protein with its ligand system, the mechanism of interaction can be explored by the dynamics analysis. However, this complex is not a receptor-ligand complex in the strict sense, as we cannot calculate the dissociation constant (KD) using a surface plasmon resonance measurement. Therefore, MD simulation is an appropriate approach to study the interactions of proteins and their ligands.

In this paper, standard MD simulations were performed to explore the interaction of PLY with VBS. On the basis of the MD simulation and the un-bond free energies, the bonding methods of VBS with PLY could be understood more deeply. Interestingly, VBS could bind to the cleft between domains 3 and 4 in PLY by making strong contact with Asp471, Asn470, Glu277, Tyr358, and Arg359. The results of the binding sites of VBS with PLY were confirmed by ligand-residue interaction decomposition using the MM-PBSA method, point mutations of residues, and a fluorescence-quenching assay. According to the above results, the results of the theoretical calculations are in good agreement with those of the experiments, and we can conclude that the binding mode of VBS with PLY can be calculated by MD simulation. The mechanism analysis of the inhibition activity depends on the dynamic analysis of the protein in the PLY/VBS complex. To our knowledge, PCA is the best tool for exploring the dynamic characteristics of a protein in a complex or unliganded state. Thus, based on our analysis of the dynamic trajectory, we can predict that the binding of VBS to PLY can cause a conformational change of domains 3 and 4 of PLY, directly leading to a decrease in the distance between domains 3 and 4. In free PLY, extended motion between domains 3 and 4 can meet the requirement of the conformational transition for PLY from the monomeric to oligomeric form. However, in the complex, the motion between domains 3 and 4 was restricted, blocking the conformational transition from the monomeric to oligomeric form. Because of that block, the lytic activity of PLY in complex is lower than that of the free protein.

The limitations of special polyvalent vaccines that prevent S. pneumoniae infection lie in their immune persistence, local reactions postinoculation, and serious reactions to vaccination (Stansfield, 1987). Based on individual contributions to pathogenesis, the most appropriate candidates for vaccines are biologic proteins that diminish the pathogenicity of the microbes, including pneumolysin (PLY), pneumococcal surface protein A, and autolysin (Jedrzejas, 2001). PLY stimulates host cell apoptosis (Braun et al., 2002), inspires complement (Paton et al., 1984), and combines with cholesterol to insert into the cell membrane and form a pore that is 350–450 Å in diameter (Gilbert et al., 1999). S. pneumoniae deleted strains of ply gene have severe attenuation in mouse models of colonization and infection (Paton et al., 1993). Particular agents have been reported to be beneficial for the treatment of diseases caused by bacterial infection, indicating that virulence factors are required for full activity and function.

High molecular weight complexes formed with PLY are indispensable for its membrane insertion, and our results demonstrate that VBS intervenes in the formation of these complexes. VBS may represent a novel PLY-specific antivirulence compound and may be beneficial in the treatment of S. pneumoniae infections when combined with regular administration of antibiotics. Although antivirulence agents have many advantageous characteristics, a great deal of additional work is required before such agents are suitable for practical clinical applications. The extensive use of these agents may lead to the development of mutations at PLY sites that eliminate VBS sensitivity; however, we do not believe that such mutations will be likely or occur at a high rate because antivirulence agents neither disrupt essential bacterial cellular functions nor exert selective pressure on pathogens.

The nucleotide sequence of PLY is highly homologous to the nucleotide sequences of other CDCs; for instance, PLY is 67% homologous to perfringolysin O of Clostridium perfringens and 76% homologous to listeriolysin O of Listeria monocytogenes. PFO, the template used for homology modeling, shares approximately 40% overall identity with the other members of the CDC family. Thus, VBS should produce similar effects on other CDCs in theory, although further research is required to elucidate the interactions of VBS with these similar proteins in practice.

Acknowledgments

Dr. David E. Briles (Departments of Microbiology, University of Alabama at Birmingham) is thanked for the generous providing of the S. pneumoniae strains D39 and strain PLN (an isogenic pneumolysin-deficient S. pneumoniae).

Authorship Contributions

Participated in research design: Zhao, Li, Wang, Deng, and Niu.

Conducted experiments: Zhao, Li, Wang, Deng, and Niu.

Performed data analysis: Zhao, Li, Guo, Liu, Deng, and Niu.

Wrote or contributed to the writing of the manuscript: Zhao, Li, Deng, and Niu.

Footnotes

Received June 30, 2015.

Accepted December 18, 2015.

Our study was supported by the National Basic Research Program of China [Grant 2013CB127205]; the National Nature Science Foundation of China [Grant 31130053]; and the National 863 program [Grant 2012AA020303].